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, 16 (1), 159

Transcriptome Analysis of Panax Vietnamensis Var. Fuscidicus Discovers Putative Ocotillol-Type Ginsenosides Biosynthesis Genes and Genetic Markers

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Transcriptome Analysis of Panax Vietnamensis Var. Fuscidicus Discovers Putative Ocotillol-Type Ginsenosides Biosynthesis Genes and Genetic Markers

Guang-Hui Zhang et al. BMC Genomics.

Abstract

Background: P. vietnamensis var. fuscidiscus, called "Yesanqi" in Chinese, is a new variety of P. vietnamensis, which was first found in Jinping County, the southern part of Yunnan Province, China. Compared with other Panax plants, this species contains higher content of ocotillol-type saponin, majonoside R2. Despite the pharmacological importance of ocotillol-type saponins, little is known about their biosynthesis in plants. Hence, P. vietnamensis var. fuscidiscus is a suitable medicinal herbal plant species to study biosynthesis of ocotillol-type saponins. In addition, the available genomic information of this important herbal plant is lacking.

Results: To investigate the P. vietnamensis var. fuscidiscus transcriptome, Illumina HiSeq™ 2000 sequencing platform was employed. We produced 114,703,210 clean reads, assembled into 126,758 unigenes, with an average length of 1,304 bp and N50 of 2,108 bp. Among these 126,758 unigenes, 85,214 unigenes (67.23%) were annotated based on the information available from the public databases. The transcripts encoding the known enzymes involved in triterpenoid saponins biosynthesis were identified in our Illumina dataset. A full-length cDNA of three Squalene epoxidase (SE) genes were obtained using reverse transcription PCR (RT-PCR) and the expression patterns of ten unigenes were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR). Furthermore, 15 candidate cytochrome P450 genes and 17 candidate UDP-glycosyltransferase genes most likely to involve in triterpenoid saponins biosynthesis pathway were discovered from transcriptome sequencing of P. vietnamensis var. fuscidiscus. We further analyzed the data and found 21,320 simple sequence repeats (SSRs), 30 primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism in 13 P. vietnamensis var. fuscidiscus accessions. Meanwhile, five major triterpene saponins in roots of P. vietnamensis var. fuscidicus were determined using high performance liquid chromatography (HPLC) and evaporative light scattering detector (ELSD).

Conclusions: The genomic resources generated from P. vietnamensis var. fuscidiscus provide new insights into the identification of putative genes involved in triterpenoid saponins biosynthesis pathway. This will facilitate our understanding of the biosynthesis of triterpenoid saponins at molecular level. The SSR markers identified and developed in this study show genetic diversity for this important crop and will contribute to marker-assisted breeding for P. vietnamensis var. fuscidiscus.

Figures

Figure 1
Figure 1
Putative pathway for triterpene saponin biosynthesis. Putative pathway for triterpene saponin biosynthesis in P. vietnamensis var. fuscidicus. Two proposed pathways (A and B) for the biosynthesis of ocotillol-type saponins, mainlymajonoside R2 in the horizontally grown rhizome (C) of P. vietnamensis var. fuscidicus (D). Enzymes found in this study are boxed. Abbreviations: AACT, acetyl-CoA acetyltransferase; β-AS, β-amyrin synthase; DMAPP, dimethylallyl diphosphate; DS, dammarenediol-II synthase; FPP, farnesyl diphosphate; FPPS, farnesyl diphosphate synthase; Glc, glucose; GPP, geranyl pyrophosphate; GGPP, geranylgeranyl diphosphate; GGPPS, geranylgeranyl pyrophosphate synthase; GT, glycosyltransferase; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; HMGR, HMG-CoA reductase; HMGS, HMG-CoA synthase; IPP, isopentenyl diphosphate; IPPI, IPP isomerase; MVD, mevalonate diphosphate decarboxylase; MVK, mevalonate kinase; P450, cytochrome P450; PMK, phosphomevalonate kinase; SE, squalene epoxidase; SS, squalene synthase.
Figure 2
Figure 2
The length distribution of contigs, unigenes and CDSs. Overview of the P. vietnamensis var. fuscidiscus transcriptome assembly and the length distribution of the CDS.
Figure 3
Figure 3
Gene Ontology classification of assembled unigenes. The unigenes were categorized into three main categories biological process, cellular component and molecular function.
Figure 4
Figure 4
COG function classification of P. vienamensis var. fuscidiscus .
Figure 5
Figure 5
Pathway assignment based on KEGG. (A) Classification based on metabolism categories; (B) Classification based on metabolism of terpenoids and polyketides.
Figure 6
Figure 6
UPGMA dendrogram of 13 accessions of P. vienamensis var. fuscidiscus. Dendrogram constructed with UPGMA clustering method among 13 different accessions of P. vienamensis var. fuscidiscus.
Figure 7
Figure 7
Phylogenetic tree of CYP450s. Phylogenetic tree of the P. vienamensis var. fuscidiscus CYP450s. Phylogenetic tree constructed based on the deduced amino acid sequences for the P. vienamensis var. fuscidiscus CYP450s (bold letters) and other plant CYP450s involved in triterpenoid biosynthesis. Protein sequences were retrieved from NCBI GenBank using the following accession numbers: Vitis vinifera VvCYP716A15, (BAJ84106.1) and VvCYP716A17 (BAJ84107.1); Medicago truncatula MtCYP716A12, (ABC59076.1), MtCYP93E2 (ABC59085), MtCYP72A63 (H1A981.1), MtCYP72A65v2, (BAL45202), MtCYP72A67v2 (BAL45203) and MtCYP72A68v2 (BAL45204), and MtCYP72A61v2 (BAL45199); Panax ginseng PgCYP716A52v2 (AFO63032.1), PgCYP716A53v2 (I7CT85.1) and PgCYP716A47 (H2DH16.2); Arabidopsis thaliana AtCYP708A2 (NP_001078732.1) and AtCYP705A5 (EFH40098); Glycyrrhiza uralensis GuCYP88D6 (B5BSX1.1), GuCYP93E3 (BAG68930) and GuCYP72A154 (H1A988.1); Avena strigosa AsCYP51H10 (ABG88965.1); Glycine max GmCYP93E1 (NP_001236154.1).
Figure 8
Figure 8
Phylogenetic tree of UGTs. Phylogenetic tree constructed based on the deduced amino acid sequences for the P. vienamensis var. fuscidiscus UGTs (bold letters) and other plant UGTs. Accession numbers in the NCBI GenBank database are as follows: Barbarea vulgaris BvUGT73C11 (AFN26667) and BvUGT73C10 (AFN26666); Arabidopsis thaliana AtUGT73C1 (NP_181213.1), AtUGT82A1 (NP_188864.1), AtUGT76B1 (NP_187742.1), AtUGT71B1 (NP_188812.1), AtUGT89B1 (NP_177529.2), AtUGT75B2 (NP_172044.1), AtUGT75C1 (NP_193146.1), AtUGT74C1 (NP_180738.1), AtUGT79B4 (Q9LJA6.1) and AtUGT79B1 (Q9LVW3.1); Solanum aculeatissimum SaGT4A (BAD89042); Medicago truncatula MtUGT73K1 (AAW56091), MtUGT73F3 (ACT34898) and MtUGT71G1 (AAW56092); Glycine max GmUGT73F4 (BAM29363); Panax notoginseng PnUGT1 (JX018210); Oryza sativa OsUGT709A4 (Q7XHR3); Saponaria vaccaria SvUGT74M1 (ABK76266); Linum usitatissimum LuUGT71A24 (AFJ52909), LuUGT82A2 (AFJ52979), LuUGT709D1 (AFJ53007), LuUGT75N1 (AFJ52962), LuUGT94G1 (AFJ53037.1), LuUGT79A3 (AFJ52973.1).
Figure 9
Figure 9
qRT-PCR analysis of unigenes involved in triterpene saponin biosynthesis. Validation of candidate P. vietnamensis var. fuscidiscus unigenes involved in triterpene saponin biosynthesis by qRT-PCR. Bars represent the mean (± SD) of four experiments.
Figure 10
Figure 10
Typical chromatograms of triterpenoid saponins in roots. Typical chromatograms of triterpenoid saponins in P. vietnamensis var. fuscidiscus roots. (A) HPLC-ELSD chromatograms of majonoside R2 in P. vietnamensis var. fuscidiscus roots; (B) HPLC-ELSD chromatograms of authentic majonoside R2. (C) HPLC chromatograms of ginsenoside Rg1, Rb1, notoginsenoside R1, and ginsenoside Rd in P. vietnamensis var. fuscidiscus roots. (D) HPLC chromatograms of ginsenoside Rg1, Rb1, notoginsenoside R1, and ginsenoside standards.

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